Two types of glial fibrillary acidic protein-positive (GFAP+) astrocytes were found in cultures of developing rat optic nerve. Type 1 astrocytes had a fibroblast-like morphology, did not bind tetanus toxin or the monoclonal antibody A2B5 (both of which bind to specific polysialogangliosides), and were stimulated to divide by an extract of bovine pituitary and by epidermal growth factor (EGF). Type 2 astrocytes had a neuron-like morphology, bound tetanus toxin and A2B5 antibody, and were not stimulated to divide by bovine pituitary extract or by EGF. Although both types of astrocytes were present in cultures of white matter, only type 1 astrocytes were found in cultures of gray matter. Astrocytes did not convert from one type to the other in culture: while many type 1 astrocytes adopted a neuron-like morphology when exposed to dibutyryl cyclic adenosine 3′:5′-monophosphate, or pituitary or brain extracts, especially in serum-free medium, such morphologically altered cells did not bind tetanus toxin or A2B5 antibody. Although small numbers of tetanus toxin-binding, A2B5+, GFAP+ cells were present in suspensions of freshly dissected, neonatal optic nerves, most of the type 2 astrocytes in cultures of such optic nerves developed from tetanus toxin-binding, A2B5+, GFAP- cells, which were induced to express GFAP by the culture conditions. Since type 2 astrocytes have a neuron-like morphology and bind tetanus toxin and A2B5 antibody, these ligands cannot be used on their own as neuron- specific markers in central nervous system cultures.